Error-pooling-based statistical methods for identifying novel temporal replication profiles of human chromosomes observed by DNA tiling arrays

Statistical analysis on tiling array data is extremely challenging due to the astronomically large number of sequence probes, high noise levels of individual probes and limited number of replicates in these data. To overcome these difficulties, we first developed statistical error estimation and weighted ANOVA modeling approaches to high-density tiling array data, especially the former based on an advanced error-pooling method to accurately obtain heterogeneous technical error of small-sample tiling array data. Based on these approaches, we analyzed the high-density tiling array data of the temporal replication patterns during cell-cycle S phase of synchronized HeLa cells on human chromosomes 21 and 22. We found many novel temporal replication patterns, identifying about 26% of over 1 million tiling array sequence probes with significant differential replication during the four 2-h time periods of S phase. Among these differentially replicated probes, 126941 sequence probes were matched to 417 known genes. The majority of these genes were found to be replicated within one or two consecutive time periods, while the others were replicated at two non-consecutive time periods. Also, coding regions found to be more differentially replicated in particular time periods than noncoding regions in the gene-poor chromosome 21 (25% differentially replicated among genic probes versus 18.6% among intergenic probes), while such a phenomenon was less prominent in gene-rich chromosome 22. A rigorous statistical testing for local proximity of differentially replicated genic and intergenic probes was performed to identify significant stretches of differentially replicated sequence regions. From this analysis, we found that adjacent genes were frequently replicated at different time periods, potentially implying the existence of quite dense replication origins. Evaluating the conditional probability significance of identified gene ontology terms on chromosomes 21 and 22, we detected some over-represented molecular functions and biological processes among these differentially replicated genes, such as the ones relevant to hydrolase, transferase and receptor-binding activities. Some of these results were confirmed showing >70% consistency with cDNA microarray data that were independently generated in parallel with the tiling arrays. Thus, our improved analysis approaches specifically designed for high-density tiling array data enabled us to reliably and sensitively identify many novel temporal replication patterns on human chromosomes.     

A cell-based nitric oxide reporter assay useful for the identification and characterization of modulators of the nitric oxide/guanosine 3',5'-cyclic monophosphate pathway

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.     

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.     

Heritability of brachydactyly type A3 in children, adolescents, and young adults from an endogamous population in eastern Nepal

Brachymesophalangia-V (BMP-V), a short and broad middle phalanx of the fifth digit, is the most common of all skeletal anomalies of the hand. When this feature appears alone, it is clinically known as brachydactyly type A3 (BDA3). A high prevalence of BDA3 has been observed among the children of the Jirel ethnic group in eastern Nepal. As part of the Jiri Growth Study, a hand-wrist radiograph is taken annually of each child to assess skeletal development. For this study the most recent radiographs of 1,357 Jirel children, adolescents, and young adults (676 boys, 681 girls), age 3-20 years, were examined for the presence or absence of BDA3, to report the prevalence and estimate the heritability of BDA3 in the Jirel population. The overall prevalence of BDA3 in this sample was 10.5% (12.9% of the males and 8.9% of the females were classified as BDA3 affected). The additive genetic heritability of BDA3 was statistically significant in this sample (h2 +/- SE = 0.87 +/- 0.16, p < 0.0001). This study is the first to estimate the prevalence and heritability of BDA3 in a large South Asian family-based sample.     

The fossil family Ameghinornithidae (Mourer-Chauviré 1981): a short synopsis

A short synopsis of the taxa included in Ameghinornithidae is presented. The rather poor fossil record suggests that these birds were (nearly?) flightless, similar to small phorusrhacids.     

Population trend over 100?years and conservation needs of breeding sandwich terns ( Sterna sandvicensis ) on the German North Sea coast

The breeding population of the sandwich tern (Sterna sandvicensis) on the German North Sea coast has undergone substantial fluctuations throughout the last 100 years. Numbers of breeding birds were fluctuating quite strongly in the first 30 years of the twentieth century. From 1930 to the mid-1950s, a relatively steady decrease occurred. A minimum was reached in 1965 with 2,243 pairs. Around 1970, numbers increased quite markedly up to the mid-1990s and reached a centurial maximum with 10,138 pairs in 1996. Most recently, the numbers have dropped to only 5,681 pairs in 2005, the lowest number over the last 30 years. Some colonies have existed over long periods, others only for short periods, with often substantial and sudden changes. The distribution at sea was studied by transect counts from ships. During the reproductive period, high totals were found between the mainland coast and the islands, up to 30 km from the outer coast/island line. The seaward extent of the sandwich tern distribution coincided quite well with the 20-m depth line. Maximum foraging ranges for single colonies were estimated to be ca. 45 km for Trischen, ca. 35 km for Norderoog and ca. 30 km for both Scharh?rn/Nigeh?rn and Juist. Overall flight ranges for all colonies were estimated at 33.8 km for 95% of the birds. Germany has a high international responsibility for the protection of this species. Only a few colonies exist every year, making this species very vulnerable to anthropogenic disturbance, pollution events and fishing activities.

L-FABP is a critical host factor for successful malaria liver stage development

The malaria parasite liver stage produces tens of thousands of red cell-infectious forms within its host hepatocyte. It is thought that the vacuole-enclosed parasite completely depends on the host cell for successful development but the molecular parasite-host cell interactions underlying this remarkable growth have remained elusive. Using a yeast two-hybrid screen and a yeast overexpression system we show that UIS3, a parasite protein essential for liver stage development, interacts directly with liver-fatty acid binding protein, L-FABP. Down-regulation of L-FABP expression in hepatocytes severely impairs parasite growth and overexpression of L-FABP promotes growth. This is the first identified direct liver stage-host cell protein interaction, providing a possible explanation for the importance of UIS3 in liver infection.     

"Cold-sensitive" mutants of the Lac repressor

Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.     

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.